Cells use microtubule-based motors for a wide range of functions. Some motors are regulated by extrinsic factors that target them to different cargoes or enhance or suppress enzymatic activity. One such factor is dynactin, a highly conserved, multiprotein complex that is ubiquitous among eukaryotes. Dynactin is best known for its contributions to cytoplasmic dynein function, but also appears to work with another motor, kinesin-2. In this proposal, I describe work that addresses two important questions: how dynactin interacts with these two motors to allow coordinated bidirectional movement and how dynactin function changes and is regulated as cells transit the cell cycle. In one series of experiments, we will explore the interactions of dynactin with dynein and kinesin-2, using a combination of standard biochemical methods and in vitro assays in which bidirectional bead and vesicle motility is reconstituted from isolated components. The structural bases of dynactin-motor interactions will be determined using electron microscopy to image complexes formed between dynactin and dynein or kinesin-2. This work will capitalize on recent advances that have been made toward elucidating the structure of dynein, as well as a recent 3D reconstruction of dynactin obtained by our group. A second line of investigation will pursue the question of how dynactin interacts with endomembranes. We have identified one dynactin subunit, p27, that appears to play a key role in membrane binding. p27 has a number of unique properties. It is present at one extreme end of the dynactin molecule where, unlike other dynactin subunits, has the capacity to be released from the rest of the dynactin structure. p27 is also phosphorylated in mitosis, which suggests that this is a mechanism for governing dynactin function and cargo choice in a cell cycle-dependent manner. Our RNAi studies indicate that p27 contributes to dynactin function in an unexpected way in mitosis. Cells lacking p27 show defects in the timing of mitotic entry and the very final events of cytokinesis, but their spindle apparatus is completely normal, unlike what is seen when dynactin function is perturbed in other ways. We believe that p27 is required for release and rebinding of dynactin/motor complexes to membranes at the start and end of mitosis, and that this dynamic cycle of dynactin recruitment is necessary for trafficking of the molecular machinery that is responsible for cytokinetic licensing. This novel hypothesis will be tested here using the techniques of live cell imaging and subcellular fractionation.